the archaeal AspRS enzyme is nondiscriminating, which means that it forms Asp-tRNAAsp and Asp-tRNAAsn, which is the intermediate in AsntRNAAsn generation by ASp-tRNAAsn amidotransferase, in contrary bacterial enzymes are discriminating ones

enzyme deficiency or mutation is involved in development of autosomal recessive disease leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation, i.e. LBSL, often manifesting in early childhood, overview

specificty of tRNA recognition by the enzyme is primarily ensured by the tRNA identity determinants, the discriminator base G37, four bases in the anticodon loop G34, U35, C36, and C38, and G10-U25 base pair in the core region of the tRNA, substrate specificity of wild-type and truncated mutant enzymes, overview

tRNA undergoes large conformational changes upon binding to the enzyme, specific charging of amino acid resdiue on tRNA, accurate recognition by the enzyme is achieved through sequence and structural signalling

the co-substrate ATP binds preferentially with three associated Mg2+ cations in an unusual, bent geometry, the Mg2+ cations play a structural role and also participate catalytically in the enzyme reaction, co-binding of the ATP-Mg3+ complex increases the Asp/Asn binding free energy difference, indicating that amino acid discrimination is substrate-assisted, molecular dynamics simulations, overview

no acylation with L-aspartate of tRNAAsn, enzyme is discriminating, because it forms Asn-tRNAAsn by direct aminoacylation, not via the intermediate of Asp-tRNAAsn, and it contains no Asp-tRNAAsn amidotransferase

no acylation with L-aspartate of tRNAAsn, enzyme is discriminating, because it forms Asn-tRNAAsn by direct aminoacylation, not via the intermediate of Asp-tRNAAsn, and it contains no Asp-tRNAAsn amidotransferase

in Thermus thermophilus Asn-tRNAAsn is formed indirectly via a two-step pathway whereby tRNAAsn is mischarged with Asp that will subsequently be amidated into Asn by an amidotransferase.The non-discriminating aspartyl-tRNA synthetase, the trimeric GatCAB tRNA-dependent amidotransferase and the tRNAAsn promoting this pathway assemble into a ribonucleoprotein particle termed transamidosome, analysis of the mechanism of Asn-tRNAAsn formation by the transamidosome, overview

in Thermus thermophilus Asn-tRNAAsn is formed indirectly via a two-step pathway whereby tRNAAsn is mischarged with Asp that will subsequently be amidated into Asn by an amidotransferase.The non-discriminating aspartyl-tRNA synthetase, the trimeric GatCAB tRNA-dependent amidotransferase and the tRNAAsn promoting this pathway assemble into a ribonucleoprotein particle termed transamidosome, analysis of the mechanism of Asn-tRNAAsn formation by the transamidosome, overview

the archaeal AspRS enzyme is nondiscriminating, which means that it forms Asp-tRNAAsp and Asp-tRNAAsn, which is the intermediate in AsntRNAAsn generation by ASp-tRNAAsn amidotransferase, in contrary bacterial enzymes are discriminating ones

enzyme deficiency or mutation is involved in development of autosomal recessive disease leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation, i.e. LBSL, often manifesting in early childhood, overview

no acylation with L-aspartate of tRNAAsn, enzyme is discriminating, because it forms Asn-tRNAAsn by direct aminoacylation, not via the intermediate of Asp-tRNAAsn, and it contains no Asp-tRNAAsn amidotransferase

no acylation with L-aspartate of tRNAAsn, enzyme is discriminating, because it forms Asn-tRNAAsn by direct aminoacylation, not via the intermediate of Asp-tRNAAsn, and it contains no Asp-tRNAAsn amidotransferase

in Thermus thermophilus Asn-tRNAAsn is formed indirectly via a two-step pathway whereby tRNAAsn is mischarged with Asp that will subsequently be amidated into Asn by an amidotransferase.The non-discriminating aspartyl-tRNA synthetase, the trimeric GatCAB tRNA-dependent amidotransferase and the tRNAAsn promoting this pathway assemble into a ribonucleoprotein particle termed transamidosome, analysis of the mechanism of Asn-tRNAAsn formation by the transamidosome, overview

in Thermus thermophilus Asn-tRNAAsn is formed indirectly via a two-step pathway whereby tRNAAsn is mischarged with Asp that will subsequently be amidated into Asn by an amidotransferase.The non-discriminating aspartyl-tRNA synthetase, the trimeric GatCAB tRNA-dependent amidotransferase and the tRNAAsn promoting this pathway assemble into a ribonucleoprotein particle termed transamidosome, analysis of the mechanism of Asn-tRNAAsn formation by the transamidosome, overview

the co-substrate ATP binds preferentially with three associated Mg2+ cations in an unusual, bent geometry, the Mg2+ cations play a structural role and also participate catalytically in the enzyme reaction, co-binding of the ATP-Mg3+ complex increases the Asp/Asn binding free energy difference

i.e. McC, upon its entry into a susceptible cell, McC is processed to release a nonhydrolyzable aspartyl-adenylate that inhibits aspartyl-tRNA synthetase, leading to the cessation of translation and cell growth. The rate-limiting step of McC processing in vitro is deformylation of the first methionine residue of McC

the N-terminal 70 amino acid residues extension protrudes from the anticodon-binding module, it is not essential for acylation activity but contains the RNA-binding motif that promotes non-specific interactions with the tRNAs, it can also interact with the 5'-end of the enzyme's mRNA

high molecular weight aminoacyl-tRNA synthetase complex contains lipid. Delipidation does not affect the size or activity of the complex, but a variety of functional and structural properties of individual synthetases in the complex are altered: sensitivity to salts plus detergents, temperature inactivation, hydrophobicity, sensitivity to protease digestion

7 different mutants, modification of crystal surfaces, investigation of crystallizability to determine the influence of surface residues and protein structure on crystal growth, packing arrangement, and quality

hanging-drop vapour-diffusion method, enzyme crystallizes either in a monoclinic or an orthorhombic habit. Minute amounts of protein impurities alter to a different extent the growth of each crystal form. The best synthetase crystals are obtained when the crystallizating solution is either enclosed in capillaries or immobilized in agarose gels

recombinant GST-tagged enzyme from Escherichia coli strain Top10 by glutathione affinity chromatography, the tag is then cleaved of by thrombin, and the enzyme is further purified by adsorption chromatography yielding an AspRS with a short additional amino acid stretch at its N-terminus

expression of His6-tagged or GST-tagged enzyme in Escherichia coli strain BL21(DE3), and expression of biotin- or His6-tagged DRS-SUMO and DRS-ubiquitin fusion proteins in Escherichia coli strain BL21(DE3), since the free DRS is expressed as an inactive and insoluble protein in Escherichia coli, SUMO is a small ubiquitin-like modifier molecule

expression of wild-type and mutant enzymes in yeast YBC-603 cells, functional overexpression of GST- and His6-tagged enzyme in Escherichia coli strain Top10. Expression in yeast cells at low copy number, since overexpression is cytotoxic, while moderate AspRS accumulation in the cell is not deleterious

the sequence corresponding to the carboxyl-terminal Ty1-tagged Tb-AspRS1 is cloned into a derivative of pLew-100 to allow tetracycline-inducible expression of the tagged protein in transgenic cell lines; the sequence corresponding to the carboxyl-terminal Ty1-tagged Tb-AspRS1 is cloned into a derivative of pLew-100 to allow tetracycline-inducible expression of the tagged protein in transgenic cell lines, into the vector pET15b for expression in Escherichia coli BL21-CodonPlus DE3-RIL cells

mutant enzyme is still able to aminoacylate the native tRNAAsp in vivo at a level sufficient to complement the defective strain CS143. 6.5fold increase in aminoacylation rate and 3fold decrease in amino acid activation reaction

thermosensitive mutant, resulting in substitution of Pro 555 by Ser. Pro555Ser lowers the stability of the functional configuration of both the acylation and the amino acid activation sites but has no significant effect on substrate binding

a naturally occuring mutation identified in patients suffering leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation. The mutant enzyme is not processed due to nontranslocation of the protein

native recombinant AspRS from rat and the N-terminal truncated derivatives Asp-DELTA20 and AspRS-DELTA36 expressed in yeast. A moderate but significant drop in affinity towards the multisynthetase complex is inferred by the removal of the N-terminal domain. This domain is absolutely required in vivo for association within the multisynthetase structure